REVIEW
published: 20 April 2015
Edited by:
Nancy Keller,
University of Wisconsin-Madison,
USA
Reviewed by:
Mikael R. Andersen,
Technical University of Denmark,
Denmark
Christopher L. Schardl,
University of Kentucky, USA
*Correspondence:
Axel A. Brakhage and
Volker Schroeckh, Department
of Molecular and Applied
Microbiology, Leibniz Institute
for Natural Product Research
and Infection Biology Hans Knll
Institute, Adolf-Reichwein-Strae 23,
07745 Jena, Germany
axel.brakhage@hki-jena.de;
volker.schroeckh@hki-jena.de
Specialty section:
This article was submitted to
Microbial Physiology and Metabolism,
a section of the journal
Frontiers in Microbiology
Received: 30 October 2014
Accepted: 26 March 2015
Published: 20 April 2015
Citation:
Netzker T, Fischer J, Weber J,
Mattern DJ, Knig CC, Valiante V,
Schroeckh V and Brakhage AA (2015)
Microbial communication leading to
the activation of silent fungal
secondary metabolite gene clusters.
Front. Microbiol. 6:299.
doi: 10.3389/fmicb.2015.00299
Frontiers in Microbiology | www.frontiersin.org
doi: 10.3389/fmicb.2015.00299
Microbial communication leading
to the activation of silent fungal
secondary metabolite gene clusters
Tina Netzker 1,2 , Juliane Fischer 1,2 , Jakob Weber 1,2 , Derek J. Mattern 1,2 , Claudia C. Knig 1,2 ,
Vito Valiante 1 , Volker Schroeckh 1* and Axel A. Brakhage 1,2*
1
Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection
Biology Hans Knll Institute, Jena, Germany, 2 Department of Microbiology and Molecular Biology, Institute of Microbiology,
Friedrich Schiller University Jena, Jena, Germany
Microorganisms form diverse multispecies communities in various ecosystems. The
high abundance of fungal and bacterial species in these consortia results in specific
communication between the microorganisms. A key role in this communication is played
by secondary metabolites (SMs), which are also called natural products. Recently,
it was shown that interspecies talk between microorganisms represents a physio-
logical trigger to activate silent gene clusters leading to the formation of novel SMs
by the involved species. This review focuses on mixed microbial cultivation, mainly
between bacteria and fungi, with a special emphasis on the induced formation of
fungal SMs in co-cultures. In addition, the role of chromatin remodeling in the induction
is examined, and methodical perspectives for the analysis of natural products are
presented. As an example for an intermicrobial interaction elucidated at the molecular
level, we discuss the specific interaction between the filamentous fungi Aspergillus
nidulans and Aspergillus fumigatus with the soil bacterium Streptomyces rapamycinicus,
which provides an excellent model system to enlighten molecular concepts behind
regulatory mechanisms and will pave the way to a novel avenue of drug discov-
ery through targeted activation of silent SM gene clusters through co-cultivations of
microorganisms.
Keywords: co-culture, secondary metabolite gene cluster activation, natural products, intermicrobial communica-
tion, posttranslational histone modifications, chromatin, acetyltransferases, mass spectrometry
Introduction
Secondary metabolites (SMs) are low-molecular-mass organic compounds that, unlike primary
metabolites, are not directly involved in growth, development or reproduction of the producing
organism. Up until 2014 170,000 natural products have been characterized from both marine
and terrestrial organisms (Seyedsayamdost and Clardy, 2014; Chapman and Hall, 2015). Microor-
ganisms are able to synthesize a large number of SMs, but the exact number is not known.
Furthermore, mining of microbial genomes revealed the presence of numerous SM gene clusters,
displaying a discrepancy between the number of putative genes involved in secondary metabolism
and the known SMs in a single microbe (Bergmann et al., 2007; Sanchez et al., 2012; Craney
et al., 2013). For example, the model fungus Aspergillus nidulans is potentially able to produce
32 polyketides, 14 non-ribosomal peptides and two indole alkaloids (Brakhage et al., 2008; Rank
et al., 2010), with little more than 50% of the produced SMs being identified. Furthermore, SMs
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Netzker et al.
FIGURE 1 | Microorganismic multispecies communities form secondary
metabolites that contribute to the stabilization/changes in these
companionships. In nature, microorganisms process signals from both abiotic
and biotic environments. The latter represent secondary metabolites or natural
can be found in diverse environments and even chemical bio-
geographic distribution maps for biomedically valuable fami-
lies of natural products in the environment have been created
(Charlop-Powers et al., 2014). A number of these compounds
have important pharmacological applications and are used as
antibiotics/antibacterial drugs (Brakhage, 2013). Unfortunately,
antibiotic resistance is spreading faster than the development of
new antibiotics. As a consequence, there is the need for a con-
stant provision of new compounds for the antibiotic development
pipeline (Bbosa et al., 2014; Nathan and Cars, 2014). This is
contrasted with a continuous rise in re-isolation of already known
natural products (Strand et al., 2014). To manage this conflict,
a more targeted natural product search is necessary. This effort
directs SM research incrementally to a deeper understanding of
the physiological relevance and ecological significance of SMs. It
is generally accepted that in nature a substantial benefit to the
SM producers must exist, simply arising from the fact that these
very energy consuming biosynthetic pathways were maintained
through evolution. An early postulated explanation for the role of
SMs in nature was its function to defend the habitats of the pro-
ducers by inhibiting the growth of its competitors (Davies, 1990;
Brakhage et al., 2005; Galn et al., 2013). Another more recent
hypothesis postulates an association between epibiotic predation
and antibiotic production due to widespread predatory abilities
in the genus Streptomyces (Kumbhar et al., 2014). At low, there-
fore non-inhibitory concentrations, such molecules are believed
to function as signaling molecules (Aminov, 2009; Andersson
and Hughes, 2014). This is supported by the assumption that
over millions of years the evolution of SMs happened because
microorganisms used them as chemical signals for communica-
tion between cells of the same species, different species (Figure 1)
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Microbial communication activates secondary metabolites
products whose formation is often suppressed in pure cultures under standard
conditions in the laboratory. Microbial mixed cultivation is therefore a suitable
means to exploit their potential for natural product discovery and to study the
molecular concepts behind the regulatory interactions.
or between host cells, e.g., as endophytes in other microorganisms
(Partida-Martinez and Hertweck, 2005) or plants (Brader et al.,
2014).
As reported above, the majority of computationally identified
SM gene clusters are silent under standard laboratory growth
conditions. Successful methods to induce the formation of new
metabolites include genetic engineering (Bergmann et al., 2007),
mutagenesis, the OSMAC approach (Bode et al., 2002) or treat-
ment with epigenetic modifiers (Cichewicz, 2010; Ntzmann
et al., 2011; Brakhage, 2013). In contrast to these classical meth-
ods, co-cultivation of bacteriabacteria, fungifungi or of bacteria
and fungi represent a naturally driven approach mimicking phys-
iological conditions, involving competition and communication.
Furthermore, co-cultures are highly relevant for drug research
because they allow not only for the identification of new com-
pounds, but can also monitor drug effects on synthetic microbial
consortia. Up to now, various co-cultivation strategies have been
applied. A summary with the focus on synthetic biology was
given by Goers et al. (2014), while successful strategies with a
special emphasis on SM formation in co-culture experiments were
recently reviewed by Bertrand et al. (2014b).
The regulatory mechanisms of SM biosynthetic gene clusters
are poorly understood. Unraveling both production conditions
and signal transduction in nature, e.g., by identifying global reg-
ulators, will help to understand their function and support new
possibilities to further explore SMs. Only a few examples on the
gene regulatory network during SM formation in co-cultures have
been reported. One is given by the specific interaction between
A. nidulans and Streptomyces rapamycinicus. Thereby, activation
of a fungal silent gene cluster and production of novel compounds
was mediated by manipulating the chromatin-based regulation
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in the eukaryotic partner by the bacteria (Schroeckh et al., 2009;
Ntzmann et al., 2011). This review focuses on the communica-
tion between microorganisms, which has led to the activation of
silent gene clusters and the formation of (novel) SMs by at least
one of the involved species. Of particular focus is the bacteria-
triggered activation of silent SM gene clusters in fungi and the
role of chromatin remodeling in SM formation. Furthermore,
methodical perspectives for the analysis of natural products are
also discussed.
Microbial Communication as an Inducer of
Silent Secondary Metabolite Gene Clusters
Microbes and their SMs are known as one of the best resources
for new drugs (Brakhage, 2013; Luo et al., 2014). Microorganisms
form diverse multispecies communities within the natural envi-
ronment. Here, they are subjected to intra- and interspecies inter-
actions, which may result in beneficial or even harmful outcomes
for the species involved. The real triggers leading to the activa-
tion of natural product biosynthesis in these communities are as
diverse as the products themselves. They range from environmen-
tal signals, such as pH, carbon and nitrogen sources, to organisms
living in the same habitat (Figure 1; Yu and Keller, 2005; Brakhage,
2013). Several recent reviews on mixed microbial cultivation
and SMs have been published (Scherlach and Hertweck, 2009;
Tarkka et al., 2009; Bertrand et al., 2014b; Marmann et al., 2014;
Schroeckh et al., 2014), which clearly support co-cultivations of
two (or even more) organisms on solid/liquid cultures as an
adequate way to trace new metabolites. Additionally, such cul-
tivations allow a tremendously enhanced production of already
known natural compounds.
Starting in 1982, when Watanabe et al. (1982) discovered
the formation of the antibacterial polyketide enacyloxin by Glu-
conobacter sp. W-315 during a co-cultivation with the fungi Neu-
rospora crassa or Aspergillus oryzae, the number of publications
dealing with mixed fermentations has drastically increased. The
vast majority have been published within the last 57 years and
nowadays co-cultivation of microbial species has turned into a key
method in the discovery of new natural products with certain rel-
evance to pharmaceutical or agricultural applications (Schroeckh
et al., 2009; Brakhage, 2013; Moody, 2014).
The typical motivation for co-cultivation experiments is the
identification of new bioactive compounds by unlocking cryp-
tic SMs present in the genomes of the microorganisms in
use. This has been shown for many microbial combinations,
i.e., bacterium-bacterium, bacterium-fungus and fungus-fungus.
Examples for SMs produced in fungus-fungus co-cultures are the
acremostatins A-C, formed by Acremonium sp. in mixed cul-
ture with Mycogone rosea (Degenkolb et al., 2002), aspergicin,
derived from a culture of two Aspergillus species (Zhu et al.,
2011) or cyclo-(l-leucyl-trans-4-hydroxy-l-prolyl-d-leucyl-trans-
4-hydroxy-l-proline), produced in the co-culture broth of two
mangrove fungi Phomopsis sp. K38 and Alternaria sp. E33 (Li
et al., 2014). Additionally, screening of fungal co-cultures in
solid media led to the identification of an unusual long-distance
growth inhibition between Trichophyton rubrum and Bionectria
ochroleuca (Bertrand et al., 2013b). Analytical methods, such as
Frontiers in Microbiology | www.frontiersin.org
Microbial communication activates secondary metabolites
LC-MS-based metabolomics (see below), identified five de novo
induced compounds, and the structure of one was successfully
achieved (4 -hydroxysulfoxy-2,2 -dimethylthielavin P).
Bacterial mixed cultures that led to the synthesis of previ-
ously unknown SMs mostly involve gram-positive bacteria, such
as streptomycetes, which form the largest genus in the actino-
mycetes order and represent an unlimited source of novel com-
pounds, including many therapeutic molecules with anti-tumor,
anti-cancer, antibiotic, and antifouling properties (Subramani and
Aalbersberg, 2012; Doroghazi et al., 2014). As reported, Strepto-
myces lividans TK23 produces a red pigment after the direct inter-
action with the mycolic acid-containing bacterium Tsukamurella
pulmonis TP-B0596 (Onaka et al., 2011). In parallel, T. pulmo-
nis TP-B0596 is also able to induce natural product synthesis
or, at least, to enhance their production in other Streptomyces
strains. Accordingly, a novel antibiotic named alchivemycin A
was isolated from the culture broth of the co-cultivation between
T. pulmonis and Streptomyces endus (Onaka et al., 2011).
Streptomycetes are not only soil microorganisms, but are also
widespread throughout marine ecosystems and have been iso-
lated from various seaweed and marine sediments. Co-cultivation
of marine streptomycetes was also successfully used to encrypt
silent gene clusters. They have also been found to represent
a promising source of antifoulants (Xu et al., 2010). Biofoul-
ing, the accumulation of microorganisms, algae and plants on
wet surfaces, is one of the most serious problems encom-
passed in various marine industries. The active antifouling diter-
pene lobocompactol was rapidly induced and isolated from the
marine actinomycete Streptomyces cinnabarinus (PK209) after co-
cultivation with the lobocompactol-resistant bacterium KNS-16
(Alteromonas sp.; Cho and Kim, 2012), leading to the isolation
of an extremely valuable compound for both marine ecology and
industry.
Induction of Fungal Silent Secondary
Metabolite Gene Clusters by
Co-Cultivation with Bacteria
In nature, interactions between bacteria and fungi are com-
monly present. Physical contact between these microorganisms
can be assumed in various environments, such as soil, food or
even patients (e.g., cystic fibrosis) where organisms can live in
close contact and compete for different resources (Frey-Klett
et al., 2011). Already in 2001, the production of pestalone,
a potent antibiotic against methicillin-resistant Staphylococcus
aureus (MRSA) and vancomycin-resistant Enterococcus faecium,
was obtained in the co-culture of a marine-derived gram-negative
bacterium of the genus Thalassopia sp. (CNJ-328) and the
marine fungus Pestalotia (Cueto et al., 2001). Although fungi-
bacteria consortia exist in both terrestrial and marine environ-
ment, the majority (>90%) of the currently known microbial
natural compounds are derived from terrestrial microorgan-
isms (Subramani and Aalbersberg, 2012). Streptomyces, Pseu-
domonas, and Bacillus are reported to be the most commonly
found bacteria in soil and the rhizosphere (Bouizgarne, 2011)
and play the most important role as fungal partners. The
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gram-positive model organism Bacillus subtilis is one of the most
frequently found microorganisms in the rhizosphere. It can also
induce SM production in fungi, as proved by the formation of
macrocarpon C, 2-(carboxymethylamino)benzoic acid and ()-
citreoisocoumarinol in Fusarium tricinctum (Ola et al., 2013).
Compared to the fungal monoculture, the production of lateropy-
rone, cyclic depsipeptides of the enniatin type, and fusaristatin
A were up to 78-fold higher (Ola et al., 2013). Also marine-
derived fungal-bacterial communities have been found to be a
promising origin of novel SMs (Marmann et al., 2014). Oh et al.
(2007) observed that co-cultivation of a marine fungus identi-
fied as Emericella parvathecia and the actinomycete Salinispora
arenicola led to a 100-fold production of emericellamides A and
B by the fungus. Both metabolites showed a slightly increased
activity against MRSA. Emericella is the teleomorph (sexual form)
of many Aspergillus spp. (Geiser, 2009). In fact, the emericellamide
biosynthetic gene cluster, which contains a PKS and a NRPS,
was also described in the model organism A. nidulans (Chiang
et al., 2008). Co-cultivation of the marine -proteobacterium
Thalassopia sp. (CNJ-328) and the fungus Libertella sp. led to the
production of libertellenones A-D by the fungus. A direct physical
contact appears to be important for libertellone production, as the
diterpenoids were neither produced in a Libertella monoculture
nor by adding supernatant or extract of the bacterial culture
(Oh et al., 2005). Libertellenones showed an increased cytotoxic
activity against human adenocarcinoma cell line (HCT-116), but
no antibiotic properties.
Aspergillus fumigatus, the most common airborne fungal
pathogen, has been reported to produce at least 226 potentially
bioactive SMs (Frisvad et al., 2009) including well studied metabo-
lites like gliotoxins, pseurotins, and fumagillins. Again, most of
the biosynthetic gene clusters are silent under laboratory condi-
tions. Zuck et al. (2011) co-cultured A. fumigatus with Strepto-
myces peucetius which led to the formation of formyl xanthocillin
analogs, named fumiformamide, and N,N -((1Z,3Z)-1,4-bis(4-
methoxyphenyl)buta-1,3-diene-2,3-diyl)diformamide. A. fumiga-
tus co-cultured with Streptomyces bullii produced ergosterol
and numerous new metabolites, including seven metabolites
of the diketopiperazine alkaloids, brevianamide F, spirotrypro-
statin A, 6-methoxy spirotryprostatin B, fumitremorgin C and
its 12,13-dihydroxy derivative, fumitremorgin B as well as ver-
ruculogen, 11-O-methylpseurotin A and a new isomer 11-O-
methylpseurotin A2 (Rateb et al., 2013). A. fumigatus is also
part of a microbial interaction in another unusual habitatcoal
mine drainages where such interactions may be helpful for
survival. Co-cultures of two coalmine drainage-derived organ-
isms, a Sphingomonas strain and an A. fumigatus strain led
to the detection of glionitrin A, which is a new diketopiper-
azine (Park et al., 2009). Glionitrin A shows significant antibi-
otic activity against both MRSA as well as increased cytotoxic
activity against four human cancer cell lines. Further poten-
tial microbial interactions were revealed in the genus Fusar-
ium, which are also filamentous fungi widely distributed in the
soil. Analysis of the interaction between Fusarium pallidoroseum
and Saccharopolyspora erythraea resulted in three new decalin-
type tetramic acid analogs related to equisetin (Whitt et al.,
2014).
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Microbial communication activates secondary metabolites
Functional Analysis of Microbial
Communication
The various examples presented above illustrate that mixed micro-
bial fermentations are an emerging field in microbiology. They
can be seen as a strategy to mimic the physiological conditions
in the different microbial consortia. The better understanding of
the native bacterial-fungal interactions will not only expand our
possibilities to identify interesting new SMs (e.g., lead structures),
but also affect our knowledge on how these consortia are struc-
tured by the signals derived from the involved species. In a recent
study it was shown how SMs contribute to the structure of micro-
bial communities (Donia et al., 2014). The biosynthetic capacity
of the human microbiome was explored by systematic analysis
of its biosynthetic gene clusters and identified the thiopeptide
lactocillin, which is produced by the vaginal commensal Lacto-
bacillus gasseri. Interestingly, lactocillin is active against several
pathogens like S. aureus and Corynebacterium aurimucosum, but
inactive against commensals thus influencing the microbial com-
position of this specific habitat. Metatranscriptomic data analysis
revealed that the corresponding thiopeptide biosynthetic gene
cluster is indeed expressed in vivo in human samples (Donia
et al., 2014). Something similar was shown in other kingdoms.
In another example the effect of SMs produced by endophytic
fungi on the cohabitating host plant was shown to provide benefits
to the host. In mixed microbial cultures the endophytic fungus,
Alternaria tenuissima, significantly increased the production of
several polyketides, including the antifungal stemphyperylenol,
which is active against another endophytic fungus, Nigrospora
sphaerica, a well-known leaf pathogen.
True symbioses between microorganisms have even shown a
fruitful source for new SMs. A very special kind of interaction
between a fungus and a bacterium is that of the zygomycete Rhizo-
pus microsporus harboring endosymbiotic bacteria of the species
Burkholderia rhizoxinica, a novel species discovered by Partida-
Martinez et al. (2007a). Together with its symbiont the fungus is an
important plant pathogen causing rice seedling blight. For more
than two decades, it was thought that the fungus produces the
causal agent for the plant disease. As shown, the endosymbiont
is the actual producer of the phytotoxin, rhizoxin, that binds to
the -tubulin of the rice plant cells and causes mitotic arrest
(Partida-Martinez and Hertweck, 2005, 2007; Partida-Martinez
et al., 2007b). This, in turn induces the typical symptoms of
swelling of the seedling tips and finally resulting in the death of the
plants offspring (Scherlach et al., 2006). Additionally, it has also
been shown that the endobacterium is obligatory for sporulation
of its host fungus (Partida-Martinez et al., 2007b). Elucidation of
the underlying molecular mechanisms of this interaction (Leone
et al., 2010) led to the discovery of self resistance mechanisms
of the fungus against the mycotoxin (Schmitt et al., 2008) and of
factors essential for symbiosis (Leone et al., 2010; Lackner et al.,
2011). Recent data revealed that a type 2 secretion system (T2SS)
is also required for the formation of the endosymbiosis between
the fungus and the endobacterium (Moebius et al., 2014). By use
of comparative proteome analysis, it was shown that chitinolytic
enzymes and chitin-binding proteins were released by the secre-
tion system of the bacterium. Further experiments (e.g., targeted
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gene-knock-outs, sporulation assays) clearly showed that a chiti-
nase is essential for the bacteria to enter the hyphae (Moebius
et al., 2014). More recently, the biosynthesis of antifungal and
antibacterial polyketides by Burkholderia gladioli in co-culture
with R. microsporus has been investigated (Ross et al., 2014).
Conditions emulating tempe bongkrek production, a type of fer-
mented soybeans made with the addition of coconut, resulted
in the formation of novel members of the enacyloxin family of
antibiotics and to enhanced production of the toxin, bongkrekic
acid, by the tempe contaminant B. gladioli.
Overall, the underlying mechanisms of SM biosynthetic gene
cluster regulation are emerging, but are still poorly under-
stood. Only few studies reported the gene regulation mechanisms
involved in SM formation during microorganism interaction. One
of them is the antibiotic concanamycin A production by Strepto-
myces halstedii. Concanamycin A alters the proteomic profile of
A. nidulans and probably plays an active role in defense-related
pathways (Melin et al., 2002). Another example, which will be
extensively described below, is the specific interaction between
A. nidulans and S. rapamycinicus. During this mutual interplay,
the activation of silent gene clusters, and subsequent production
of novel compounds, is transduced by affecting the chromatin-
based regulation in the eukaryotic partner (Schroeckh et al., 2009;
Ntzmann et al., 2011).
The Interaction of Aspergillus
with Streptomyces rapamycinicus
It was discovered that the intimate physical contact of A. nidu-
lans with a distinct soil-dwelling bacterium, S. rapamycinicus,
identified from a collection of 58 species of actinomycetes, led
to the selective activation of silent PKS and NRPS gene clus-
ters in the fungus (Schroeckh et al., 2009). One induced cryptic
PKS gene encodes the long sought-after orsellinic acid synthase,
thus the corresponding cluster was named the ors gene cluster.
In addition to this archetypal polyketide orsellinic acid, three
derivatives (lecanoric acid and two cathepsin K inhibitors F-
9775A and F-9775B) were produced by A. nidulans. Lecanoric
acid is a typical lichen metabolite that is usually found in a
fungal/bacterial mutualism (Stocker-Worgotter, 2008), and thus
likely plays a role in microbial communication. Indeed, the induc-
ing bacterium was not affected by lecanoric acid. As mentioned
above, a physical contact between both partners is needed for
the activation of this silent gene cluster (Scherlach and Hertweck,
2009; Schroeckh et al., 2009). It is conceivable that a symbiotic
relation between the fungus and the bacterium exists to defend
against other microorganisms. One explanation on how the bac-
terium can trigger SM formation in Aspergillus would have been
that rapamycin produced by the streptomycete could activate the
cluster, either via the inhibition of the TOR pathway (Fitzgibbon
et al., 2005) or due to its more general antifungal activity. Alter-
natively, another bacterial metabolite, the fungistatic antibiotic
trichostatin A (TSA), which is produced by Streptomyces hygro-
scopicus could be responsible via its respective histone deacetylase
(HDAC) inhibiting activity (Tsuji et al., 1976; Kouraklis and
Theocharis, 2002). However, neither the addition of rapamycin
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Microbial communication activates secondary metabolites
nor TSA led to the activation of the ors gene cluster, therefore
making both compounds unlikely to play a role in the interaction.
When S. rapamycinicus was co-cultivated with A. fumigatus, this
fungus also displayed an altered SM profile showing a group of
similar new SMs (Knig et al., 2013). In a microarray approach, a
SM gene cluster was identified that was up-regulated only in the
co-culture. Deletion of the PKS of the identified cluster, correlated
with the lack of the corresponding natural products. Two metabo-
lites of this group were isolated and named fumicycline A and B
and the corresponding PKS was designated as FccA. It was shown
that again a direct physical contact was necessary to induce the
fcc gene cluster. An ortholog of the fcc gene cluster was identified
in Neosartorya fischeri (Chooi et al., 2013). Overexpression of the
transcription factor gene of the cluster led to the production of
neosartoricins. These metabolites demonstrate high similarity to
fumicyclines and showed T-cell antiproliferative activity, suggest-
ing a physiological role as an immunosuppressive agent (Chooi
et al., 2013).
Studies of various chemical inhibitors led to the striking finding
that the interaction between S. rapamycinicus and A. nidulans
relies largely on the activity of chromatin remodelers. Supple-
mentation of the co-culture with a TSA-like HDAC inhibitor,
suberoylanilide hydroxamic acid (SAHA), and with the histone
acetylase (HAT) inhibitor anacardic acid led to the activation and
inhibition of the transcription of the ors gene cluster, respectively.
These findings indicated that chromatin remodeling can play an
essential role in the regulation of SM clusters and that the targeted
activation or inactivation of the respective chromatin modifiers
can alter the SM production of the fungus. Ntzmann et al. (2011)
demonstrated that acetylation plays an essential role for mediating
the interaction. Therefore, a comprehensive deletion library of all
putative HATs in A. nidulans was generated and systematically
screened for the ability of mutants to activate the ors gene cluster
during co-incubation with S. rapamycinicus (Ntzmann et al.,
2011). Thereby, the HAT GcnE was identified as being essential
for the cluster induction. This HAT is the catalytic subunit of the
Saga/Ada complex (see Figure 2), a conserved multi-subunit com-
plex also found in other eukaryotic organisms (Baker and Grant,
2007; Govind et al., 2007). Furthermore, it was shown that the
acetylation of histone H3 lysines 9 and 14 is needed for the onset
of the ors gene cluster transcription and product formation (Ntz-
mann et al., 2013). However, SAGA not only seemed to play a role
during the interaction with S. rapamycinicus, but also for the reg-
ulation of other well-known natural products such as penicillin,
sterigmatocystin and terrequinone A in A. nidulans (Ntzmann
et al., 2011, 2013). Due to the intimate contact of S. rapamycinicus
with A. nidulans the question arose whether there is a common
mechanism by which the bacterium might interact with other
members of the Aspergillus family, e.g., with A. fumigatus. It is
possible to postulate different ways that can lead to the activation
of such clusters in the fungus. As shown in Figure 2, an unknown,
possibly membrane-bound compound or a protein can modulate
the Saga/Ada complex directly (Figures 2a,b). Alternatively, the
signal could be induced either by the physical contact between
the two organisms (Figure 2c), or by a protein or compound
secreted by the bacterium, and specifically sensed by receptors of
A. nidulans (Figure 2d). These components need to be specific for
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FIGURE 2 | Model of the interaction between A. nidulans and S.
rapamycinicus. The figure presents hypotheses about different stimuli that
could be responsible for the activation of SM gene clusters during the
interaction between A. nidulans and S. rapamycinicus. The signal that finally
results in the Saga/Ada complex activation could derive from at least five
possible events: possibly membrane-bound compounds (a), or peptides (b),
S. rapamycinicus and must not be found in other actinomycetes.
Convincible is also that the recognition of a fungal surface protein
by the streptomycete, could directly lead to the activation of a
signaling cascade triggering the SAGA complex (Figure 2e). In
the interaction of A. nidulans with S. rapamycinicus the ors gene
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Microbial communication activates secondary metabolites
could reach the cytosol of the fungus and indirectly activate the Saga/Ada
complex. Alternatively, a specific fungal receptors could recognize either the
attachment of the bacterium (c), or compounds secreted during the interaction
(d). A further hypothesis could be that fungal surface proteins could recognize
the streptomycete directly triggering a signaling cascade (e). The internal signal
that should lead to the activation of the Saga/Ada complex is unknown.
cluster regulation relies largely on the activity of GcnE and its
acetylation activity of lysine 9 and 14 at histone H3. This in turn is
induced upon physical contact with the bacterium leaving room
for speculation on the key influence of the streptomycete on the
fungus. Regarding the signaling pathway behind this interaction,
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it is known that LaeA as a global SM regulator has no influence on
GcnE and therefore on histone H3 acetylation (Ntzmann et al.,
2011). This means that there must be an alternative pathway and
a transcriptional regulator responsible for the recruitment of the
HAT and the multi-subunit complex, SAGA, to the respective loci.
For some clusters, such as penicillin, it has already been shown
that some general regulators are required like the major pH regu-
lator PacC, which activates penicillin biosynthesis at alkaline pH
in A. nidulans, or the CCAAT binding complex (Tilburn et al.,
1995; Litzka et al., 1998; Then Bergh and Brakhage, 1998). For
the ors gene cluster, however, the key regulators still remain to be
discovered.
Modulation of Gene Expression
by Interaction Partner Induces Chromatin
Remodeling
The impact of chromatin remodeling on the communication
between organisms or the control of host gene expression has
gained attention in the last years. There are several examples of
bacterial pathogens interfering with the host regulatory system of
gene expression. Fewer are known about the regulatory mecha-
nisms of interactions involving fungi, especially when it comes
to those leading to the expression of SM gene clusters. However,
some light has been shed on the impact of chromatin remodeling
on natural product regulation in fungi. By now, a variety of
chromatin modifiers have been discovered, which regulate SM
biosynthesis in filamentous fungi (Gacek and Strauss, 2012). Most
knowledge so far has been gained on acetyltransferases (HATs),
which are grouped in diverse families, comprising amongst oth-
ers the MYST-family, p300/CBP-family, and the Gcn5-related
acetyltransferases (GNATs) (Carrozza et al., 2003). The latter
includes Gcn5, the catalytic subunit of the SAGA complex, also
referred to as GcnE in Aspergillus species. As mentioned before,
a distinct deletion mutant of the knock-out library of HATs in
A. nidulans led to an altered SM biosynthesis pattern. Hence,
it was speculated that the systematic screening of the deletion
library allows for the identification of novel metabolites. Indeed, a
drastically altered metabolic profile was detected in the nnaB
(nidulans N-acetyltransferase B) deletion mutant. Aside from a
number of orsellinic acid derivatives, there was also a new class
of compounds identified as pheofungins, which are heterocyclic
molecules with cytotoxic activity (Scherlach et al., 2011). HAT
modification led the production of SMs also in other fungi. The
aflatoxin biosynthetic gene cluster in Aspergillus parasiticus was
also shown to be co-regulated by a MYST-type family member of
the HATs. Furthermore, the transcription of the aflatoxin cluster
genes coincides with the acetylation of histone H4, showing that
a HAT is involved in the regulation of this cluster (Roze et al.,
2007, 2011). Soukup et al. (2012) obtained similar results by
overexpressing esaA, a gene encoded for a MYST-type HAT. The
overexpression of this gene affected the production of penicillin,
sterigmatocystin, terrequinone A, and the ors gene clusters in
A. nidulans (Soukup et al., 2012).
The example of S. rapamycinicus, which mediates cluster reg-
ulation in A. nidulans via an increased histone acetylation upon
Frontiers in Microbiology | www.frontiersin.org
Microbial communication activates secondary metabolites
contact, shows how bacteria can interfere with the eukaryotic his-
tone modification system (epigenetic regulation). Other bacteria
were also reported to have a similar impact on eukaryotic cells.
Listeria monocytogenes, the producer of the toxin listeriolysin O,
is a bacterium causing foodborne infections leading to sepsis, mis-
carriages during pregnancy, and meningitis, and is largely found
in immunocompromised patients. Hamon et al. (2007) were able
to show that listeriolysin O caused a dramatic modulation of the
host gene expression. This was caused by deacetylation of histone
H4 but also drastic dephosphorylation of serine 10 on H3 thus
leading to a downregulation of substantial immunity factors in the
host cells. Similar observations have been made for Clostridium
perfringens and Streptococcus pneumoniae, the producers of per-
fringolysin and pneumolysin, respectively (Maekita et al., 2006;
Hamon et al., 2007). Both toxins also led to dephosphorylation of
the hosts chromatin. Thus, different toxins secreted by bacteria
appear to manipulate and control chromatin remodeling and
thereby transcription of targeted genes of eukaryotic hosts.
The so-called erasers of acetylation are the HDACs that fulfill
the opposing reaction of the HATs by removing the acetyl group
from lysine residues of the histone proteins. HdaA, a class II
HDAC, was one of the first discovered to play a role in SM
cluster regulation. The deletion of hdaA in A. nidulans not only
led to reduced growth of the fungus during oxidative stress, but
also resulted in a higher production of SMs such as penicillin,
sterigmatocystin, and terrequinone A (Tribus et al., 2005; Shwab
et al., 2007). Consistently, HdaA had a significant impact on
SM produced in A. fumigatus, such as fumitremorgin B, pseu-
rotin, and gliotoxin. Interestingly, however, was the finding that
gliotoxin production was down-regulated upon deletion of hdaA
in A. fumigatus (Shwab et al., 2007; Lee et al., 2009). Garcia-Garcia
et al. (2009) were able to connect the activity of the human HDAC1
with the infection process of Anaplasma phagocytophilum in THP-
1 cells (granulocyte model). Hereafter, the infection process led to
an increased activation of HDAC1 leading to a reduced histone
H3 acetylation and to the silencing of host defense genes. In
accordance, the inhibition of HDAC1 by siRNA led to a significant
drop of the bacterial load. This shows that the epigenetic control
of the host cell by the bacterium promotes the disease by increased
survival of the pathogen (Garcia-Garcia et al., 2009).
Besides the widely studied acetylation of histones, there is a
multitude of other chromatin modifications which have been
found to regulate expression of SM gene clusters (Strahl and
Allis, 2000). Methylation of lysine is regarded as one of the most
complex modifications found so far with diverse impacts on
gene transcription depending on its conformation (Rolando et al.,
2013). Reyes-Dominguez et al. (2010) showed that upon growth
arrest the methylation of lysine 9 was subsequently reduced, but
affected only genes located inside the sterigmatocystin cluster,
leading to its activation. Furthermore, H3 K9 methylation marks
were associated with heterochromatin protein A (HepA), a pro-
tein responsible for heterochromatin formation. Consistently, the
deletion of HepA led to the activation of the stc gene cluster
(Reyes-Dominguez et al., 2010; Brakhage, 2013). However, the
combination of both the hepA and the laeA deletions reduced the
sterigmatocystin production to wild-type levels (Shaaban et al.,
2010). The global SM regulator LaeA was indirectly found to
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Netzker et al.
be involved in histone methylation by influencing the methy-
lation of H3 K9 and the occupancy of the respective locus by
HepA. The deletion of this gene was also found to constrain
the expression of the prominent natural product gene clusters
penicillin, sterigmatocystin and the cholesterol lowering agent
lovastatin (Bok and Keller, 2004; Reyes-Dominguez et al., 2010).
In another study, the heterochromatin protein, HP1 of the fungus
Leptosphaeria maculans, could be implicated in the pathogenic-
ity process responsible for plant infection. The fungus harbors
effector genes with low expression during axenic cultivation,
while being highly transcribed upon co-cultivation with plants.
In an infection model with Brassica napus the effect of histone
H3K9me3 on the respective effector genes was investigated. HP1
as well as the DNA-methylase DIM-5 were silenced by RNAi and
analyzed concerning the transcription level of the effector genes
in axenic cultures. Interestingly, the effector genes were actively
transcribed in the mutant strains outside of the co-cultivation
leading to the conclusion that HP1 as well as DIM-5 must be
somehow involved in the repression of the effector genes during
the non-infective life cycle (Soyer et al., 2014). Additionally, in
a symbiotic interaction of the endophyte Epichlo festucae and
Lolium perenne, the fungus produced ergot alkaloids and lolitrems
when cohabitating with a plant. Production of these bioprotec-
tive substances was repressed during axenic cultures. Comparing
levels of H3K9me3 and H3K27me3 between co-cultivation and
non-symbiotic cultivation of the fungus, the methylation marks
were reduced upon growth in the plant. Furthermore, the deletion
of the responsible methyltransferases ClrD and EzhB resulted in
an activation of the alkaloid and lolitrem gene clusters also in the
axenic cultures of the fungus (Chujo and Scott, 2014).
Methylation of lysines 4 at histone H3 by CclA was found to
be important for SM biosynthesis as well as for conidiation in A.
nidulans and A. fumigatus (Palmer et al., 2008, 2013; Bok et al.,
2009). The deletion of the respective genetic locus in A. nidulans
not only led to the production of F-9775A and F-9775B, which are
also produced upon contact with the bacterium S. rapamycinicus,
but led also to the activation of a novel monodictyphenon gene
cluster. Another very interesting study was published by Rolando
et al. (2013). They showed that pathogens can also introduce, so
far, unknown modifications on host nucleosomes and thereby
influence gene expression. In their study they elegantly revealed
that Legionella pneumophila is able to tri-methylate lysine 14 on
histone H3 of its host by a factor called RomA. This protein
is a SET-domain containing methyltransferase which is secreted
by Legionella during the infection process. Genome-wide ChIP
analysis showed that approximately 4870 promoters were target
of this modification by RomA (Rolando et al., 2013). The cause
for this drastic modulation of the host genome by Legionella is
not fully understood yet. One possible explanation might be that
the switch to a methylated histone leads to down-regulation of
the target genes due to a mutual exclusion of the acetylated lysine
14, which was found to coincide with actively transcribed genes
(Cheung et al., 2000).
In summary, the impact of post-translational regulation on SM
cluster expression and the interaction of organisms have revealed
their great potential for future studies in natural product research.
Prokaryotes are able to modify their hosts gene expression in
Frontiers in Microbiology | www.frontiersin.org
Microbial communication activates secondary metabolites
multiple ways. Bacterial toxins were shown to be useful tools
during the infection process by reducing levels of acetylation and
phosphorylation of histones and thereby influencing the expres-
sion of their target genes. Often, chromatin modifying complexes
are mediators of those interactions, specifically targeting host
defense genes and modulating their expression. Interestingly, this
is not only achieved by host derived remodelers but also by pro-
teins introduced by the interaction partner itself, which in turn
can lead to unknown modification on the host genome. Taken
together, recent studies have shown the great potential of bacteria
and fungi to modulate gene expression of organisms during co-
cultivation experiments. Seeing this, it is convincible that the
investigation at the molecular basis of multispecies interaction has
great potential. The more we understand about communication
between species, the better we can trigger the discovering of
unknown natural products in microorganisms.
Perspectives for the Analysis of Natural
Products
In the search for SMs in co-cultivations, one must also deter-
mine which analytical method to use for the detection of these
compounds. This topic has already been extensively reviewed by
other groups (Scherlach and Hertweck, 2009; Tarkka et al., 2009;
Bertrand et al., 2014b; Marmann et al., 2014; Schroeckh et al.,
2014), but the most interesting new studies will be covered here.
Thus far, the methods for natural product analysis have ranged
from simply the extraction of co-cultures in liquid/solid media
to the use of quite novel techniques such as imaging/real-time
mass spectrometry that can be carried out in solid-state cultures.
The former technique has shown to be of value resulting in the
discovery of many new SMs or in the study of the regulation of
different products, which cannot be found in monocultures. This
technique commonly entails the extraction of the natural products
from the culture broth, which are then subjected to a form of
liquid chromatography-mass spectrometry (LC-MS). In a further
step, potential new SMs can be purified and isolated for structural
elucidation by nuclear magnetic resonance (NMR) spectroscopy
(Figure 3). This workflow was applied to the discovery of new
SMs from the co-cultivation of S. rapamycinicus with A. nidulans
and A. fumigatus, respectively. In both cases new fungal prod-
ucts were detected by LC-MS when the streptomycete was added
(see above). Other examples for LC-MS detection of co-culture-
derived products are the new antibiotic alchivemycin A in S. endus
by the mycolic acid-containing bacteria T. pulmonis (Onaka et al.,
2011; see above), as well as the co-culture of Streptomyces coeli-
color with myxobacterium Corallococcus coralloides, where the
streptomycete increased the production of the biologically active
compound undecylprodigiosin 60-fold (Schberle et al., 2014).
Because of its potential for the discovery of new natural products,
there is also a need for high-throughput methods to encompass
large-scale co-cultivations. This question was addressed for the
study of more than 600 different fungal strains. With the help of
automated data analysis, new molecular masses were observed
which were not found in natural product databases (Bertrand
et al., 2013a). In a further example of high-throughput screening,
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Netzker et al.
FIGURE 3 | Proceeding of natural product analysis from microbial
co-cultivations. Microbial communities are co-cultured in flasks (static or
planktonic) or on plates (solid-state culture). Primary analysis of natural products
fungal co-cultures were cultivated with very small culture vol-
umes. A big advantage of small culture volumes is that sample
preparation can be completed in less time and the number of dif-
ferent cultures can be increased (Bertrand et al., 2014a). These are
just few examples to show that LC-MS analysis of co-cultivations
can be a very practical tool, and because of the constant problem to
obtain enough product for structure elucidation, scale-up of these
cultures can usually be accomplished.
Besides the well-established exploration of co-cultures for new
bioactive compounds by LCMS, there has also been advance-
ments in the field of imaging and real-time mass spectrometry
(Fang and Dorrestein, 2014), where metabolites can be detected
by their spatial distribution. Imaging mass spectrometry (IMS)
has, for the most part, been associated with matrix-assisted laser
desorption/ionization (MALDI), which is then coupled to a mass
spectrometer, for which images can be produced depicting the
spatial organization of natural products (Cornett et al., 2007;
Esquenazi et al., 2009; Watrous and Dorrestein, 2011; Bouslimani
et al., 2014; Shih et al., 2014). This technique has shown to be
useful in observing the role of natural products in the interaction
between different microorganisms, such as A. fumigatus with
Pseudomonas aeruginosa (Moree et al., 2012), B. subtilis with S.
coelicolor (Yang et al., 2009), B. subtilis with S. aureus (Gonzalez
et al., 2011), and the cannibalism of B. subtilis (Liu et al., 2010),
just to name a few. Additionally, two promising examples, which
aided in the discovery of novel natural products by IMS will
Frontiers in Microbiology | www.frontiersin.org
Microbial communication activates secondary metabolites
via extraction and LCMS methods or directly evaluated by imaging mass
spectrometry based methods. Subsequent structure identification of unknown
masses can be conducted using NMR techniques.
be discussed. The first demonstrated that the infection of the
button mushroom, Agaricus bisporus, with the soft rot-causing
bacterium Janthinobacterium agaricidamnosum and the brown
blotch disease-causing bacterium Pseudomonas tolaasii. J. agarici-
damnosum infected mushrooms revealed the presence of a novel
virulence factor, jagaricin when analyzed by MALDI-MS at the
sites of infection. This substance was shown to play an important
role in soft rot of mushrooms and also appeared to be a potent
antifungal (Graupner et al., 2012). The second example, also using
IMS, investigated the infection of A. bisporus with P. tolaasii and
could show that the tolasin metabolites, which were observed at
the site of infection, are responsible for this disease (Scherlach
et al., 2013).
Similarly, a more recently developed technique, real-time mass
spectrometry, encompassing the techniques of desorption elec-
trospray ionization (DESI) or nanospray desorption electrospray
ionization (nanoDESI), is also a sufficient option in detecting
natural products in co-cultivations. An advantage of this method
compared to MALDI, is that it does not depend on the formation
of the matrix and also has little to no sample preparation. Mea-
surements can be taken directly from the plate and can also be
used for IMS. Moreover, the method is usually termed ambient
mass spectrometry because ionization takes place at atmospheric
conditions and room temperature. For further information the
following reviews are recommended (Bouslimani et al., 2014; Fang
and Dorrestein, 2014; Hsu and Dorrestein, 2015). Furthermore,
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Netzker et al.
Watrous et al. (2013) have also shown a methods paper using
nanoDESI IMS with little sample preparation of different micro-
bial monocultures and co-cultures directly from a Petri dish.
nanoDESI IMS has even led to the discovery of new desferriox-
amine derivatives in co-cultures of S. coelicolor with other actino-
mycetes (Traxler et al., 2013). The use of IMS for the detection
of natural products is an ever-evolving field and new techniques
are constantly being discovered and older techniques optimized.
One recent ionization method, direct analysis in real time mass
spectrometry (DART-MS), could also be used in studying the role
of SMs in co-culture (Gross, 2014).
Conclusion
Secondary metabolites are of major interest due to their appli-
cability as therapeutic agents. To satisfy the constant need for
new SMs, and to come up against the continuous emerging of
bacterial resistant strains, it would be advantageous to understand
the SMs physiological relevance and their ecological significance.
In this context, mixed microbial cultivations have become a
powerful method to induce previously unexpressed biosynthetic
pathways, leading to the production and identification of new
SMs (Schroeckh et al., 2009; Bertrand et al., 2014b; Marmann
et al., 2014). A greater understanding of the underlying molecular
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Conflict of Interest Statement: The Guest Associate Editor Nancy Keller declares
that, despite having collaborated with author Axel A. Brakhage, the review process
was handled objectively and no conflict of interest exists. The authors declare
that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.
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